Background The purpose of the study was to evaluate the diagnostic value of gamma-glutamyl transpeptidase to alkaline phosphatase ratio (GAPR) combined with gamma-glutamyl transpeptidase to aspartate aminotransferase ratio (GAR) and alanine aminotransferase to aspartate aminotransferase ratio (AAR) in alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). Methods A total of 925 AFP-negative patients, including 235 HCC patients, 213 chronic hepatitis (CH) patients, and 218 liver cirrhosis (LC) patients, as well as 259 healthy controls were enrolled in this study. The differences of laboratory parameters and clinical characteristics were analyzed by Mann-Whitney U or Kruskal-Wallis H-test. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of GAPR, GAR, and AAR in AFP-negative HCC (AFP-NHCC) patients. Results GAPR, GAR, and AAR were important parameters closely related to AFP-NHCC. The combination of GAPR, GAR, and AAR was most effective in differentiating AFP-NHCC group from control group (AUC = 0.875), AFP-negative CH group (AUC = 0.733), and AFP-negative LC group (AUC = 0.713). GAPR combined with GAR and AAR exhibited a larger AUC than single ratio or pairwise combination for distinguishing AFP-NHCC group with TNM?stage, BCLC stage A, and tumor size less than 3 cm. The diagnostic value of GAPR combined with GAR and AAR was higher in AFP-NHCC and was also reflected in the TNM stage, Barcelona Clinic Liver Cancer (BCLC) stage and tumor size. Conclusions GAPR combined with GAR and AAR were effective diagnostic markers of AFP-NHCC, especially in patients with good liver function, early stage or small size.